Temperature-responsive polymer particles in protein separation applications

Abstract

The present invention relates to a method for isolating proteins from a solution containing the proteins. The invention also relates to a method for the chromatographic separation of proteins. The present invention also relates to crosslinked hydroxylic polymer particles functionalized with temperature-responsive copolymer, and to methods of preparing such particles.

The invention claimed is: 
     
       1. A method for isolating proteins from a solution containing the proteins the method including:
 a. providing polymeric ion exchange resins comprising hydroxylic polymer particles functionalized with a temperature-responsive copolymer, including a proportion of ionizable chemical groups, wherein the copolymer is propagated on the hydroxylic polymer particles and the polymeric ion exchange resins do not comprise a silica matrix; 
 b. contacting the solution containing the proteins with the polymeric ion exchange resins at a temperature between 30° C. and 80° C. to facilitate retention of the proteins by the crosslinked polymer particles; 
 c. replacing the solution containing the protein with a rinse solution; 
 d. replacing the rinse solution with a release solution, wherein the temperature of the release solution is lower than the temperature at which the solution containing the proteins was contacted with the crosslinked polymer particles and is effective for releasing the proteins from the crosslinked polymer particles; and 
 e. isolating the release solution containing the protein. 
 
     
     
       2. A method according to  claim 1 , wherein the protein solution and the rinse solution are at a temperature between 30° C. and 60° C. and the release solution is at a temperature between 0° C. and 20° C. 
     
     
       3. A method according to  claim 1 , wherein the release solution contains an ionic solute which is an alkali metal halide or an alkali earth metal halide. 
     
     
       4. A method according to  claim 1 , wherein the temperature-responsive copolymer includes:
 a. monomer units providing temperature-responsive properties to the copolymer; and 
 b. monomer units providing ionizable chemical groups to the copolymer. 
 
     
     
       5. A method according to  claim 1 , wherein the monomer units providing temperature-responsive properties to the copolymer are selected from the group consisting of N-isopropylacrylamide units, vinyl methyl ether units or N-vinylcaprolactam units and the monomer units providing ionizable chemical groups are selected from the group consisting of acrylic acid units, methacrylic acid units, ethacrylic acid units, sodium 2-acrylamido-2-methylpropanesulfonate units, sodium 3-acrylamido-3-methylbutanoate units, (3-acrylamidopropyl)tri ethyl ammonium chloride units, N 1 N-dimethylaminopropylacrylamide units, N,N-dimethylaminoethyl methacrylate units, N,N-dimethylaminoethyl acrylate units, and 4-vinylbenzyltrimethylammonium chloride units. 
     
     
       6. A method according to  claim 1 , wherein the temperature-responsive copolymer further includes at least one bi-functional monomer unit selected from the group consisting of ethylene glycol dimethacrylate units, 1,4-butanediol dimethacrylate units, 1,6-hexanediol dimethacrylate units, ethylene glycol diacrylate units, 1,4-butanediol diacrylate units, 1,6-hexanediol diacrylate units, and N,N-methylenebisacrylamide units. 
     
     
       7. A method according to  claim 1 , wherein the temperature-responsive copolymer further includes at least one additional monomer unit selected from the group consisting of methyl acrylate units, ethyl acrylate units, propyl acrylate units, butyl acrylate units, methyl methacrylate units, ethyl methacrylate units, propyl methacrylate units, N-isopropylmethacrylamide units, butyl methacrylate units, N-tertbutylacrylamide units, N-N-dimethylacrylamide units, N,N-diethylacrylamide units, and N-phenylacrylamide units. 
     
     
       8. A method according to  claim 1 , wherein the crosslinked polymer particles are selected from the group consisting of crosslinked agarose particles, crosslinked cellulose particles, hydrophilic crosslinked vinyl polymer particles, and methacrylate based polymeric resin particles. 
     
     
       9. A method according to  claim 1 , wherein steps (a)-(c) occur in a chromatographic column having an inlet and an outlet wherein the crosslinked polymer particles functionalized with temperature-responsive copolymer are included in the path between the inlet and outlet. 
     
     
       10. A method according to  claim 9 , wherein the solution containing the proteins, the rinse solution and the release solution are introduced sequentially through the inlet and collected from the outlet. 
     
     
       11. A method according to  claim 1 , wherein the protein is selected from the group consisting of lactoferrin, lactoperoxidase, papain and cytochrome C.